Normal Murine Embryonic Fibroblasts Research Articles

Hydrophilic platinum(II) complexes containing 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine: Spectroscopic and in vitro cytotoxic characterization

Mononuclear platinum(II) complexes with 5,7-methyl-1,2,4-triazolo[1,5-a]pyrimidine (dmtp) of the general formula cis-[Pt(X)2(dmtp)2], where X - acetate (1), trichloroacetate (2), trifluoroacetate (3), nitrate (4) have been synthesized and characterized by multinuclear magnetic resonance (1H, 13C, 15N, 195Pt) and infrared spectroscopy methods. Spectroscopic parameters indicated the presence of the PtO2N2 chromophore system with two monodentate N(3)-bonded 5,7-methyl-1,2,4-triazolo[1,5-a]pyrimidines, and two monodentate O-donor ligands (carboxylate or nitrate).A cytotoxicity assay of hydrophilic platinum(II) complexes (1–4) against three human tumour cell lines A549 (non-small cell lung carcinoma), T47D (breast cancer), HT-29 (colon adenocarcinoma), and normal murine embryonic fibroblast cells (BALB/3T3) was performed.Considering the cytotoxic parameters of the studied complexes, the best in vitro results against the human breast tumour cells (T47D) were found for cis-[Pt(OOCCCl3)2(dmtp)2] although all the tested complexes (except complex (4)) exhibited moderate in vitro activity.

Platinum(II) Complexes with Bulky Disubstitute Triazolopyrimidines as Promising Materials for Anticancer Agents.

Herein, we present dicarboxylate platinum(II) complexes of the general formula [Pt(mal)(DMSO)(L)] and [Pt(CBDC)(DMSO)(L)], where L is dbtp 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine) or ibmtp (7-isobutyl-5-methyl-1,2,4- triazolo[1,5-a]pyrimidine), as prospective prodrugs. The platinum(II) complexes were synthesized in a one-pot reaction between cis-[PtCl2(DMSO)2], silver malonate or silver cyclobutane-1,1-dicarboxylate and triazolopyrimidines. All platinum(II) compounds were characterized by FT-IR, and 1H, 13C, 15N and 195Pt NMR; and their square planar geometries with one monodentate N(3)-bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine, one S-bonded molecule of dimethyl sulfoxide and one O,O-chelating malonato (1, 2) or O,O-chelating cyclobutane-1,1-dicarboxylato (3, 4) was determined. Additionally, [Pt(CBDC)(dbtp)(DMSO)] (3) exhibited (i) substantial in vitro cytotoxicity against the lung adenocarcinoma epithelial cell line (A549) (IC50 = 5.00 µM) and the cisplatin-resistant human ductal breast epithelial tumor cell line (T47D) (IC50 = 6.60 µM); and (ii) definitely exhibited low toxicity against normal murine embryonic fibroblast cells (BALB/3T3).

A novel machine learning based approach for iPS progenitor cell identification.

Identification of induced pluripotent stem (iPS) progenitor cells, the iPS forming cells in early stage of reprogramming, could provide valuable information for studying the origin and underlying mechanism of iPS cells. However, it is very difficult to identify experimentally since there are no biomarkers known for early progenitor cells, and only about 6 days after reprogramming initiation, iPS cells can be experimentally determined via fluorescent probes. What is more, the ratio of progenitor cells during early reprograming period is below 5%, which is too low to capture experimentally in the early stage. In this paper, we propose a novel computational approach for the identification of iPS progenitor cells based on machine learning and microscopic image analysis. Firstly, we record the reprogramming process using a live cell imaging system after 48 hours of infection with retroviruses expressing Oct4, Sox2 and Klf4, later iPS progenitor cells and normal murine embryonic fibroblasts (MEFs) within 3 to 5 days after infection are labeled by retrospectively tracing the time-lapse microscopic image. We then calculate 11 types of cell morphological and motion features such as area, speed, etc., and select best time windows for modeling and perform feature selection. Finally, a prediction model using XGBoost is built based on the selected six types of features and best time windows. Our model allows several missing values/frames in the sample datasets, thus it is applicable to a wide range of scenarios. Cross-validation, holdout validation and independent test experiments show that the minimum precision is above 52%, that is, the ratio of predicted progenitor cells within 3 to 5 days after viral infection is above 52%. The results also confirm that the morphology and motion pattern of iPS progenitor cells is different from that of normal MEFs, which helps with the machine learning methods for iPS progenitor cell identification.

In search of new anticancer drug – Dimethylsulfoxide ruthenium(III) complex with bulky triazolopyrimidine derivative and preliminary studies towards understanding the mode of action

By reacting of [H(dmso)2]trans-[RuCl4(dmso)2] with 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) in molar ratio of M:L = 1:2 new ruthenium(III) complex mer,cis-[RuCl3(dbtp)2(dmso)] has been prepared and structurally characterized by X-ray, IR and EPR spectroscopies. The crystal structure of the Ru(III) complex showed slightly distorted octahedral geometry with unique cis positioned monodentate dbtp ligands bound to the ruthenium(III) ion through the N3 nitrogen atom. In vitro toxicity assay proves that slightly lipophilic the mer,cis-[RuCl3(dbtp)2(dmso)] (logP = 0.80) is 5-fold less toxic against non-tumorigenic human epithelial cell line (MCF-10A) and normal murine embryonic fibroblast cells (BALB/3T3) than cisplatin. Preliminary studies towards understanding the mode of action suggested that activation by reduction, electrostatic interactions of the mer,cis-[RuCl3(dbtp)2(dmso)] with CT-DNA and selective delivery to cells by apotransferrin might be relevant for the biological properties of the complex.

AMPK 2 Suppresses Murine Embryonic Fibroblast Transformation and Tumorigenesis

AMP-activated kinase (AMPK) is a key metabolic sensor and stress signaling kinase. AMPK activity is known to suppress anabolic processes such as protein and lipid biosynthesis and promote energy-producing pathways including fatty acid oxidation, resulting in increased cellular energy. In addition, AMPK localizes to centrosomes during cell division, plays a role in cellular polarization, and directly targets p53, affecting apoptosis. Two distinct catalytic AMPKα isoforms exist: α1 and α2. Multiple reports indicate that both common and distinct functions exist for each of the 2 α isoforms. AMPK activation has been shown to repress tumor growth, and it has been suggested that AMPK may function as a metabolic tumor suppressor. To evaluate the potential role of each of the AMPKα isoforms in modulating cellular transformation, susceptibility to Ras-induced transformation was evaluated in normal murine embryonic fibroblasts (MEFs) obtained from genetically deleted AMPKα1- or AMPKα2-null mice. This study demonstrated that while AMPKα1 is the dominant AMPK isoform expressed in MEFs, only the AMPKα2-null MEFs displayed increased susceptibility to H-RasV12 transformation in vitro and tumorigenesis in vivo. Conversely, AMPKα1-null MEFs, which demonstrated compensation with increased expression of AMPKα2, displayed minimal transformation susceptibility, decreased cell survival, decreased cell proliferation, and increased apoptosis. Finally, this study demonstrates that AMPKα2 was selectively responsible for targeting p53, thus contributing to the suppression of transformation and tumorigenic mechanisms.

Cytoprotective Autophagy Induction by Imatinib Mesylate In Non-BCR-ABL Expressing Cells

AbstractAbstract 4937Autophagy is an intracellular degradation system that delivers cytoplasmic constitutions to the lysosome. Intracellular proteins and organelle are sequestered by the autophagosomes, then delivered to the lysosome and degraded. It has been reported that combining imatinib mesylate (IM) with an autophagy inhibitor such as chloroquine resulted in enhanced elimination of CML clones in vitro and in vivo (Bellodi C et al. J. Clin. Invest. 2009). We here report that IM induces the cytoprotective effect in non-BCR-ABL expressing cells along with autophagosome formation.Treatment with IM resulted in an increased viable cell number of non-BCR-ABL expressing leukemia cell lines by inhibiting spontaneous apoptosis. IM did not show any effects on cell-cycle progression. When HL-60 cells were cultured in the presence of 3–30 μM of IM for 24–96 hr, up to a 1.6-fold increase in the viable cell number was observed in a dose-dependent manner. Furthermore, when HL-60 cells were cultured either under serum depletion or at lower concentrations of FBS, the cytoprotection by IM became more pronounced as compared with the cells cultured with 10% FBS. As well as in BCR-ABL expressing CML cell lines, electron microscopy revealed an increased autophagosomes after IM treatment in non-BCR-ABL expressing cells. Cytoprotection with autophagosome formation by IM was observed in various leukemia and cancer cell lines as well as normal murine embryonic fibroblasts (MEFs). IM attenuated the cytotoxic effect of cytosine arabinoside and bortezomib in leukemia cells.Inhibition of autophagy by knockdown of LC3 gene using shRNA in HL-60 as well as complete knockout of atg5 in the Tet-off atg5-/- MEF system attenuated the cytoprotective effect of IM. This indicates that the effect is at least in part dependent on autophagy. However, the cytoprotection by IM was not medicated through supression of ROS production via “mitophagy” or reduction of ER stress via “ribophagy”. The cytoprotection with autophagy was still detectable in the HL-60 cells after knockdown of c-abl gene and in an ABL deficient-HL-60 cell line. This indicates that the proapoptotic function of ABL kinase is not involved. In addition, this effect was still observed in the presence of cycloheximide, suggesting that the effect is not mediated through translation process.Although the target tyrosine kinase(s) of IM remains unclear, our data provide novel therapeutic possibilities of targeting tyrosine kinase(s) for induction of cytoprotective autophagy. Disclosures:No relevant conflicts of interest to declare.

Cytoprotective effect of imatinib mesylate in non-BCR-ABL-expressing cells along with autophagosome formation

Treatment with imatinib mesylate (IM) results in an increased viable cell number of non-BCR-ABL-expressing cell lines by inhibiting spontaneous apoptosis. Electron microscopy revealed an increase of autophagosomes in response to IM. IM attenuated the cytotoxic effect of cytosine arabinoside, as well as inhibiting cell death with serum-deprived culture. Cytoprotection with autophagosome formation by IM was observed in various leukemia and cancer cell lines as well as normal murine embryonic fibroblasts (MEFs). Complete inhibition of autophagy by knockdown of atg5 in the Tet-off atg5−/− MEF system attenuated the cytoprotective effect of IM, indicating that the effect is partially dependent on autophagy. However, cytoprotection by IM was not mediated through suppression of ROS production via mitophagy, ER stress via ribophagy, or proapoptotic function of ABL kinase. Although the target tyrosine kinase(s) of IM remains unclear, our data provide novel therapeutic possibilities of using IM for cytoprotection.

Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity.

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-beta gene. The resultant virus (VSV-IFNbeta) was successfully propagated in cells not receptive to murine IFN-alpha/beta and expressed high levels of functional heterologous IFN-beta. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNbeta was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-beta, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNbeta grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-beta gene (VSV-hIFNbeta) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNbeta was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNbeta retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.

Lipoprotein Lipase Binds to Low Density Lipoprotein Receptors and Induces Receptor-mediated Catabolism of Very Low Density Lipoproteins in Vitro

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of plasma triglycerides, promotes binding and catabolism of triglyceride-rich lipoproteins by various cultured cells. Recent studies demonstrate that LPL binds to three members of the low density lipoprotein (LDL) receptor family, including the LDL receptor-related protein (LRP), GP330/LRP-2, and very low density lipoprotein (VLDL) receptors and induces receptor-mediated lipoprotein catabolism. We show here that LDL receptors also bind LPL and mediate LPL-dependent catabolism of large VLDL with Sf 100-400. Up-regulation of LDL receptors by lovastatin treatment of normal human foreskin fibroblasts (FSF cells) resulted in an increase in LPL-induced VLDL binding and catabolism to a level that was 10-15-fold greater than in LDL receptor-negative fibroblasts, despite similar LRP activity in both cell lines. This indicates that the contribution of LRP to LPL-dependent degradation of VLDL is small when LDL receptors are maximally up-regulated. Furthermore studies in LRP-deficient murine embryonic fibroblasts showed that the level of LPL-dependent degradation of VLDL was similar to that in normal murine embryonic fibroblasts. LPL also promoted the internalization of protein-free triglyceride emulsions; lovastatin-treatment resulted in 2-fold higher uptake in FSF cells, indicating that LPL itself could bind to LDL receptors. However, the lower induction of emulsion catabolism as compared with native VLDL suggests that LPL-induced catabolism via LDL receptors is only partially dependent on receptor binding by LPL and instead is primarily due to activation of apolipoproteins such as apoE. A fusion protein between glutathione S-transferase and the catalytically inactive carboxyl-terminal domain of LPL (GST-LPLC) also induced binding and catabolism of VLDL. However GST-LPLC was not as active as native LPL, indicating that lipolysis is required for a maximal LPL effect. Mutations of critical tryptophan residues in GST-LPLC that abolished binding to VLDL converted the protein to an inhibitor of lipoprotein binding to LDL receptors. In solid-phase assays using immobilized receptors, LDL receptors bound to LPL in a dose-dependent manner. Both LPL and GST-LPLC promoted binding of VLDL to LDL receptor-coated wells. These results indicate that LPL binds to LDL receptors and suggest that the carboxyl-terminal domain of LPL contributes to this interaction.

Non-immune non-activated chicken macrophage destroy murine fibroblast

Non-immune non-activated chicken bone marrow-derived macrophages (BM MØ) killed murine embryonic fibroblasts in vitro. Following precultivation for 10–35 days chicken BM MØ had the capacity to destroy normal murine embryonic fibroblasts at effector: target ratios of 10:1 to 1:1. Optimal killing was observed following cocultivation of MØ and fibroblasts for 48–72 hrs. Addition of LPS neither initiated nor potentiated MØ-mediated killing. This study demonstrates that chicken MØ have the capacity to destroy, in vitro, cells of phylogenetically distant species, similarly to the ability of murine MØ to kill chicken fibroblasts. It is suggested that vertebrate MØ xenolytic potential is analagous to the capacity of invertebrate phagocytes to destroy xenografts.